The field of biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing. Relevant biomolecules that have been the focus of such screenings include chemical libraries, nucleic acid libraries, and peptide libraries in search for molecules that either inhibit or augment the biological activity of identified target molecules. With particular regard to peptide libraries, the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. However, one particular problem with peptide libraries is the difficulty of assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
The green fluorescent protein from Aequorea victoria is a 238 amino acid protein displaying autofluorescent properties. The crystal structure of the protein and several point mutants has been solved (Ormo, M. et al. (1996) Science 273: 1392-95; Yang. F. et al. (1996) Nature Biotechnol. 14: 1246-51). The fluorophore, consisting of a modified tripeptide, is buried inside a relatively rigid β-can structure, where it is almost completely protected from solvent access. The protein fluorescence is sensitive to a number of point mutations (Phillips, G. N. (1997) Curr. Opin. Struct. Biol. 7: 821-27). Since any disruption of the structure allowing solvent access to the fluorophoric tripeptide result in fluorescence quenching, the fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein.
Green fluorescent proteins have been cloned from Renilla reniformis, Renilla muelleri, and Ptilosarcus gurneyi (see WO 99/49019, hereby expressly incorporated by reference). The core chromophore sequence of the Renilla reniformis GFP and Ptilosarcus GFPs is different from Aequorea victoria GFP, and the Renilla forms have fluorescence characteristics with higher molar absorbance coefficient and narrower absorption/emission spectra as compared to Aequorea victoria GFP (Ward, W. W. et al. (1979) J. Biol. Chem. 254: 781-88). The lack of significant homology to Aequorea victoria GFP suggests that Renilla and Ptilosarcus forms provide important alternatives to the extensively exploited Aequorea victoria GFP.
Abedi et al (Nucleic Acids Res. 26, 623-30, 1998) have inserted peptides between residues contained in several GFP loops. Inserts of the short sequence LEEFGS (SEQ ID NO:1) between adjacent residues at 10 internal insertion sites were tried. Of these, inserts at three sites, between residues 157-158, 172-173 and 194-195 gave fluorescence of at least 1% of that of wild type GFP. Only inserts between residues 157-158 and 172-173 had fluorescence of at least 10% of wild type GFP. When—SAG-random 20 mer-GAS-peptide sequences were inserted at different sites internal to GFP, only two sites gave mean fluorescence intensities of 2% or more of the GFP-random peptide sequences 10-fold above background fluorescence. These sites were insertions between residues 157-158 and 172-173.
It is an object of the invention to provide compositions of fusion constructs of library peptides with scaffold proteins, comprising for example detectable proteins such as GFP, and methods of using such constructs in screening of peptide libraries.